Structural resolution of a small organic molecule by serial X-ray free-electron laser and electron crystallography

Structure analysis of small crystals is important in areas ranging from synthetic organic chemistry to pharmaceutical and material sciences, as many compounds do not yield large crystals. Here we present the detailed characterization of the structure of an organic molecule, rhodamine-6G, determined at a resolution of 0.82 Å by an X-ray free-electron laser (XFEL). Direct comparison of this structure with that obtained by electron crystallography from the same sample batch of microcrystals shows that both methods can accurately distinguish the position of some of the hydrogen atoms, depending on the type of chemical bond in which they are involved. Variations in the distances measured by XFEL and electron diffraction reflect the expected differences in X-ray and electron scatterings. The reliability for atomic coordinates was found to be better with XFEL, but the electron beam showed a higher sensitivity to charges.


Supplementary Discussion
Comparison of rhodamine-6g structures In the SX, rt-ED, cryo-ED structures, the plane of the stacked rings is approximately perpendicular to the plane of the phenolic ring (C17 to C22), which would impede resonance between rings and hinder electron transfer 52,53 .The configuration of the dimers in this orthorhombic crystal is nearly identical to that in an iodide salt crystal (CCDC#129329; Supplementary Table 2), but different from the triclinic crystal structure with chlorine determined previously by XRD from a single crystal, in which the planes of the two rings are oblique to each other rather than normal 23 (Extended Data Fig. 1, f-h).This particular dye exhibits a tendency to aggregate at higher concentrations, and hence, the effects of dimerization have been studied with respect to its photochemical properties 54,55 .A similar orientation of the H-type dimer to the SX and ED orthorhombic crystal structures was also proposed in a recent theoretical calculation 55 .However, the oxycarboxyl plane is flipped, and the configuration is different from that in the experimentally obtained structures here.The monomer conformation of the SX, rt-ED and cryo-ED structures likewise differs from the organic-solvent and metal complex structures of rhodamine-6g or its derivative (CCDC#1298021; CCDC#674356; Supplementary Table 2).

Further consideration on variations in hydrogen densities
In addition to the polarity of bonding and the motion of hydrogen atoms indicated in the main text, other factors such as smearing by truncation of information in reciprocal space, isotropic and anisotropic atomic displacement parameters, and anharmonicity along the bonding direction 18 should also be considered for quantification of the chemical characteristics at each site through electron-density and Coulomb-potential maps.The requirement for deconvolution of these factors, however, is beyond the precision limits of the current SX and ED experiments.
Practically, observations like those in this study could follow hybrid-analyses of XRD, ND and/or theoretical calculations 18,24,56 .Well-resolved hydrogen atoms, as seen in aromatic and amide groups, would be suitable candidates for further analyses.

Comparison in the electron density dimension
Residual densities in the difference maps appear noisier for the ED data (Fig. 3d, f) than for the SX data (Fig. 3b), which may represent that assignment of electron scattering factors are suboptimal as mentioned.We then converted the rt-ED experimental structure factors to X-ray structure factors by the Mott-Bethe formula 49,50 instead of view in the Coulomb-potential dimension.A difference Fourier electron density map between the converted data and calculation with neutral charges now reveals interpretable features, excess of electron on an oxygen atom but deficiency on carbon and some hydrogen atoms (Supplementary Fig. 2).This must reflect electronegativity of each atom, indicating that ED data contain information of charges.In contrast, it is known to be difficult to express electron scattering by target molecules including charges with simple theoretical scattering curves in CBED, and so structure factors are optimized against experimental CBED patterns independently of the theoretical curves 57,58,59 .This kind of treatment will need further investigation for application to 3D ED.

Supplementary Table 1 | Geometries of the atomic models of rhodamine-6g
Values in parentheses are estimated standard uncertainties derived from the full-matrix least squares refinement.
a Defined as a cross-section cutting through the center of the xanthene ring along O13 -C6 and perpendicular to the plane of the xanthene ring (see main text and Extended Data Fig. 7).

Supplementary Table 5 | Geometry of hydrogen bonding around amides
a The disposition of the three atoms is not allowed for hydrogen-bond formation.
Crystallographic structuresORTEP drawing and our responses to the A/B-level alerts of rhodamine-6g with 'SX' data (CCDC# 2119567) _vrf_PLAT919_Rh6G_SX_1 ; PROBLEM: Reflection # Likely Affected by the Beamstop ... 1 Check RESPONSE: The beamstop did not affect on reflections in our experimental setups.; _vrf_PLAT934_Rh6G_SX_1 ; PROBLEM: Number of (Iobs-Icalc)/Sigma(W) > 10 Outliers .. 9 Check RESPONSE: There are currently no certain reason to remove these outliers in this new method (serial data collection scheme) and we keep them.; ORTEP drawing and our responses to the A/B-level alerts of rhodamine-6g with 'rt-ED' data (CCDC# 2180418) _vrf_RINTA01_Rh6G_rtED_0 ; PROBLEM: The value of Rint is greater than 0.25 RESPONSE: The value is calculated from the multiple crystals and relatively high Rint values are common for electron diffraction due to dynamical scattering.; _vrf_SHFSU01_Rh6G_rtED_0 ; PROBLEM: The absolute value of parameter shift to su ratio > 0.10 RESPONSE: Relatively high su values are common for electron diffraction due to dynamical scattering.Additional refinement cycles did not improve the value.; _vrf_PLAT020_Rh6G_rtED_0 ; PROBLEM: The Value of Rint is Greater Than 0.12 ......... 0.620 Report RESPONSE: The value is calculated from the multiple crystals and relatively high Rint values are common for electron diffraction due to dynamical scattering.; _vrf_PLAT770_Rh6G_rtED_0 ; PROBLEM: Suspect C-H Bond in CIF: C2_1 --H15_1 .2.05 Ang.RESPONSE: Not C-H Bond.This would be wrongly detected because the N-H bond is elonged to fit in Coulomb potential density.; _vrf_PLAT080_Rh6G_rtED_0 ; PROBLEM: Maximum Shift/Error ............................ 0.14 Why ?RESPONSE: Relatively high su values are common for electron diffraction due to dynamical scattering.Additional refinement cycles did not improve the value.; _vrf_PLAT082_Rh6G_rtED_0 ; PROBLEM: High R1 Value .................................. 0.16 Report RESPONSE: Relatively high R-values are common for electron diffraction due to dynamical scattering.Additional refinement cycles did not improve the value.; _vrf_PLAT084_Rh6G_rtED_0 ; PROBLEM: High wR2 Value (i.e.> 0.25) ................... 0.38 Report RESPONSE: Relatively high R-values are common for electron diffraction due to dynamical scattering.Additional refinement cycles did not improve the value.; _vrf_PLAT340_Rh6G_rtED_0 ; PROBLEM: Low Bond Precision on C-C Bonds ............... 0.01226 Ang.RESPONSE: Relatively low bond precision are common for electron diffraction due to dynamical scattering.Additional refinement cycles did not improve the value.; ORTEP drawing and our responses to the A/B-level alerts of rhodamine-6g with 'cryo-ED' data (CCDC# 2180417) _vrf_RINTA01_Rh6G_cryoED_0 ; PROBLEM: The value of Rint is greater than 0.25 RESPONSE: The value is calculated from the multiple crystals and relatively high Rint values are common for electron diffraction due to dynamical scattering.; _vrf_SHFSU01_Rh6G_cryoED_0 ; PROBLEM: The absolute value of parameter shift to su ratio > 0.20 RESPONSE: Relatively high su values are common for electron diffraction due to dynamical scattering.Additional refinement cycles did not converge.; _vrf_PLAT020_Rh6G_cryoED_0 ; PROBLEM: The Value of Rint is Greater Than 0.12 ......... 1.081 Report RESPONSE: The value is calculated from the multiple crystals and relatively high Rint values are common for electron diffraction due to dynamical scattering.; _vrf_PLAT080_Rh6G_cryoED_0 ; PROBLEM: Maximum Shift/Error ..

Table 2 | Crystal systems of rhodamine-6g compounds in Cambridge Structural Database and in this work.
a Atomic composition is shown for one rhodamine-6g molecule.